Correcting Image Defects

Most images include some imperfections, the result of the inherent limitations in illumination, optics, camera or the specimen itself. Many of these can be improved by processing.

Color Spaces and Color Filtering - Most images include some imperfections, the result of the inherent limitations in illumination, optics, camera or the specimen itself. Many of these can be improved by processing. If color images are being acquired, it is first of all important to understand something about color representations.

Reducing Noise - The word “noise” is used to describe portions of an image (or any electronic signal) that do not represent the original scene, but instead arise in the detector or electronics, or in some other way (e.g., flickering fluorescent lights, printing technology, etc.). There are at least three distinct categories of noise that are dealt with in different ways: random or speckle variations, shot noise, and interference or periodic noise.

Nonuniform Illumination - Adjusting a light microscope to have uniform illumination across the entire picture width can be difficult. Scanning electron microscopes often show shading that darkens portions of the image because of detector location and specimen tilt. Lighting a copy stand uniformly is particularly hard. And many optical systems cause vignetting because less light from the scene reaches the corners of the sensor. Add to those causes any variation in the sample (nonuniform thickness, a surface that isn’t perfectly flat, etc.) and the result is an image in which the same objects have different brightnesses depending on where they lie. Such variations make subsequent analysis very difficult and should be corrected.

Expanding Image Contrast - While making adjustments to brightness and contrast is often the first thing users try to do to digitized images, that is a poor strategy. Problems of color adjustment, nonuniform illumination, and noise should be corrected first as shown above. That will make those procedures more consistent and will also allow for a greater range of contrast adjustment afterwards.

Focus Limitations - It is, of course, always desirable to acquire images in perfect optical focus. But sometimes it isn’t possible, and software must be called in to help out. One situation arises when the optics have insufficient depth-of-field to provide sharp focus for the entire scene or sample. Historically, microscopists would adjust focus up and down through the necessary range and then make a sketch showing the entire specimen, based on their mental compilation of in-focus information. Deconvolution of image blur may also be applied if necessary.

Interpolation - In the previous example of merging images to construct an extended focus composite, it is important for the images to be aligned. This is also the case for arithmetic image combinations such as tracking motion by subtracting one image from another, removing background, or Boolean combinations that will be described later on. Sometimes, for instance with the confocal light microscope, a sequence of aligned images can be captured directly. In other cases, such as typical serial section work, computer processing is needed to perform the alignment. In general this may require rotating an image and perhaps stretching to compensate for distortions in cutting.